Limulus Amebocyte Lysate (LAL) derived from the blood cells of the American horseshoe crabs react with endotoxin (Levin et al. (1964) Bull. Johns Hopkins Hosp. 115:265-274) or (1→3)-β-D-glucan (Kakinuma et al. (1981) Biochem. Biophys. Res. Commun. 101:434-439; Morita et al. (1981) FEBS Lett. 129:318-321), forming a gel. Endotoxin and β-glucan trigger two distinct LAL pathways, activation of either of which leads to gelation (Iwanaga et al. (1992) Thromb. Res. 68:1-32). Endotoxin is a cell wall component of Gram-negative bacteria, and is pyrogenic, mitogenic, and potentially lethally toxic. Accordingly, accurate and reliable detection of endotoxin is important for confirming the safety of parenteral drugs. LAL assays are the accepted standard for endotoxin detection (see, for example, the United States Pharmacopeia “Bacterial Endotoxins Test”).
Because LAL reacts with either endotoxin or β-glucan, knowing which of the two activators is present in a sample is not necessarily straightforward. β-glucan is commonly found in fungi, yeast, algae, and plants, and causes false positives in Bacterial Endotoxin Tests. β-glucan contamination has been reported in parenteral drugs, such as blood products (Ikemura et al. (1989) J. Clin. Microbiol. 27(9):1965-1968), and medical devices, such as hemodialyzers (Peason et al. (1984) Artif. Organs 8:291-298). The detection of β-glucan contamination in parenteral drugs and medical devices is useful to avoid unexpected rejection of a product that should not be rejected (Cooper et al. (1997) J. Parenter. Sci. Technol. 51:2-6).
β-glucan detection can also be used on human blood samples to assist in the diagnosis of deep mycosis (Obayashi et al. (1995) Lancet 345:17-20; Mori et al. (1997) Eur. J. Clin. Chem. Clin. Biochem. 35:553-560; Odabasi et al. (2004) Clin. Infect. Dis. 39:199-205; Ostrosky-Zeichner et al. (2005) Clin. Infect. Dis. 41:654-659). The FDA and the Japanese government have each approved such assays as clinical diagnostic methods.
The challenge has been to develop reliable, cost-effective assays for β-glucan detection that can distinguish the presence of β-glucan from the presence of endotoxin. Several processes for increasing the β-glucan specificity of LAL have been reported.
Fractionation methods (Obayashi et al. (1985) Clin. Chim. Acta 149:55-65; Kitagawa et al. (1991) J. Chromatography 567:267-273; and U.S. Pat. No. 5,681,710) are based on removing the endotoxin sensitive factor (“Factor C”) from LAL by column chromatography, or by separation steps between solid absorbents and LAL. The column chromatography and separation steps must be performed aseptically. Thus, these systems can require expensive instruments and significant efforts to prevent contamination of the system with endotoxin or β-glucan.
Another approach suppresses endotoxin activity in samples using endotoxin-neutralizing peptides (U.S. Pat. Nos. 5,616,557; 5,622,833; and 5,750,500). Efforts to suppress endotoxin activity can be affected by high amounts of endotoxin, and require the addition of anti-endotoxin substances that may be expensive and contaminated with endotoxin or β-glucan.
U.S. Pat. No. 5,571,683 adds an endotoxin neutralizing peptide to a β-glucan assay with LAL. This method requires the purification of an endotoxin neutralizing peptide from the blood of horseshoe crabs. The purification can be expensive, and avoiding contamination with endotoxin and β-glucan during the purification can be difficult.
Similarly, U.S. Pat. No. 5,266,461 adds an antibody against Factor C. This method requires preparation, synthesis and purification of antibody, again adding expense and the risk of contamination with endotoxin or β-glucan during the purification process.
Thus, despite the efforts over the past two decades to develop β-glucan assays, there remains a need for a simple method for preparing LAL with a reduced sensitivity to endotoxin.